Fluorescence techniques will be used to examine the molecular mechanism of regulation of smooth muscle myosin function. Fluorescent and/or chromophoric compounds will be used to label the myosin active site, the two different kinds of light chains, and a specific site on the rod portion of the molecule. These labels on myosin will be used to probe conformational transitions which may be important to the mechanism of smooth muscle regulation by light chain phosphorylation. Distances between these probes and other reporter groups at specific sites on actin will be measured by fluorescence energy transfer techniques. The effects of phosphorylation of the regulatory light chains by either myosin light chain kinase or protein kinase C upon these distances will be examined. These probes will also be used in experiments designed to assess the polarity, size, shape and flexibility of the myosin molecules under conditions where the 6 S (extended) to 10 S (folded) forms of myosin are known to exist. Using these approaches, the hypothesis that the head-tail junction of the myosin molecule plays a key role in the mechanism of regulation of smooth muscle myosin will be tested. This information will be useful to determine how the phosphorylation dependent conformational changes observed in vitro relate to physiologically important structural changes within myosin filaments in vivo.